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1. Generate mutants by mutagenesis of seeds Use a genetic background with lots of known polymorphisms compared to other genotypes. Availability of polymorphicmarkers for mapping.
2. Select mutants with phenotypes of interest eg. Hairless or Glabra
3. Clean up mutant genotype by backcross to wild typeall F1 will be heterozygous, mutant phenotype willbe either dominant or recessive.
4. Allelism tests with mutants that look similar
5. Select F2’s that are homozygous for the mutation again.These can be used to map the location of the mutation.
Map-based cloning of interesting genesIn a model organism
GLABRA1 (GL1)
• Involved in trichome initiation– Transcription factor– Expressed in leaf primordia early trichome
initiation• gl1 mutants result in near complete loss of trichome
initiation
Wild type gl1 mutant
Scanning EM picture of Arabidopsis top leaf surface with trichomes
Parents: Col-0 gl1/gl1 X La-er GL1/GL1 genotypes
F1 is self fertilized – all chromosomes recombine in meiosis
F2 plants – recombined chromosomes segregate
How to do this with an organism that cannot fertilize itself, like a mouse?
Mapping Cross
Mapping an Arabidopsis gene
Analyze segregation data in an F2 population.
Both chromosomes have had the opportunity to become recombined in the F1 parent
To avoid confusion, we focus on one locus of interest.
We chose individuals that are homozygous for one allele at that locus, eg. a clear phenotype.
Closely linked markers will also be homozygous in the chosen individuals. As markers are farther away on the chromosome, more of the individuals will have two different alleles for the marker genes.
Interval mapping:
Identify markers linked to the gene of interestthat define an interval on a chromosome.
Markers that define major regions of the Arabidopsis chromosomes
Marker 1 from a previous year. The first lane is the glabra mutant (Columbia), the second lane is a mixture of DNA from lane 1 and lane 3 The third lane is LandsbergThe rest are DNA from F2 plants
If marker is unlinked to GL1, we expect ¼ L/L, ½ L/C, ¼ C/C
F2s are selected as homozygous recessivegl1/gl1 by phenotype
eg. Scored for 5 markers
marker 1 2 3 4 5
1, 2 are not linked to GL1 13: 25: 12 C/C:C/L:L/LMap distance is calculated as #recombinant alleles/total X 100 cM 50% of alleles are C and 50% are L. Therefore the map distance from GL1 to 1 is 50 cm.
Marker 3 is linked to GL1. 46 C/C: 4 C/L: 0 L/L
The map distance from GL1 to 3 is 4/100 X 100 cM or 4 cM.
We established that gl1 is on Chromosome 3.
What do we do next?
We can only find a locus by identification of recombination events on either side.
Identify 2 markers on Chromosome 3 that must be on either side of gl1
Markers 3, 4 and 5 are linked to GL1on chromosome 3
We need to find another markeron the opposite side of marker 3 to define
the interval that contains GL1.
gl1?
3
4
5
gl1?
There is a recombination event between marker 3 and gl1
Is gl1 closer to the centromere or the telomere? Marker 5 is centromericMarker 4 is telomeric
Plants 3, 4, 5 and 10 are useful to identify flanking markers
Plants 3, 4 and 5 have recombination points within the interval that defines the location of GL1
They will be useful for further mapping
Plant 3 is C/L at 3L/L at 4 andC/C at 5
Plant 4 is C/C at 3C/C at 4 andC/Lat 5
Plant 5 is C/L at 3C/L at 4 andC/C at 5
Col-0
La-er
gl1
3
4
5
Plant 4
Plant 3
Plant 5
gl1
3
4
5
gl1
3
4
5
gl1
3
4
5
gl1
3
4
5
gl1
3
4
5
We will screen more F2 plants to identifythose with a recombination on either side of our chosen interval to narrow in on the location of the GL1 gene.
We will analyze the alleles of new markers located between marker 3 and marker 5.
We will only analyze DNA from plants heterozygousat either marker 3 or marker 5.
Identify more plants with recombination in the interval
From comparison of genome sequence to a recombination map made by Lister and Dean,we learned that Arabidopsis has approximately 250 kb per map unit. That represents about 100 genes.
For convenience, we aim for map resolution of 0.1 map units, which should represent 25-100 kb and hopefully 10-20 genes.
50-100 kb is the normal insert size for BAC clones.
In order to get to map resolution of 0.1 map units we screen at least 1000 F2 plants (2000 chromosomes)
Recombination frequency is calculated:
Number of recombinants/number of chromosomes,
1 recombinant chromosome/2000 chromosomes= 0.05 map units.
We can only find a locus by identification of recombination eventson either side.
Therefore, with 2000 chromosomes we should find one marker 0.05 cM to the right of GL1 and another marker 0.05 cm to the left.
An interval of 0.1 map units between the two closest markersis the best we measure.
If we want better resolution, we need more markers (which we have) and more potential recombinant chromosomes from F2 plants.
How to decide the number of F2’s to examine?
MDF20 MYN21
BAC T22A15 100 kb insert
BAC sequence gives us a list of genes. ~20 in Arabidopsis.
GenBank annotation gives us a list of predicted genes for each BAC from our ordered library.
Potential functions of the predicted genes are defined by homology to other proteins.
Candidate genes can be chosen by predicted function and expression pattern.
Once we have defined 2 markers flanking our interval that are physically close enough, we start sequence analysis for point mutations.
Go to TAIR for GL1 marker on AGI map click clone, then look at gene annotation
Candidate genes can be PCR amplified from the mutant and the sequence can be compared to wild type.
When a mutation is identified, we call that a candidate gene.
Transform mutant plant with the wild type candidate gene for complementation.
Alternatively, the entire BAC can be broken into subclones. Each subclone can be used to transform the mutant plant.
If the BAC is made with wild type DNA, subclones with the correctgene in them will complement the mutation.
Grant et al 1995 Science 269;843-846.
Final confirmation
• Sequence mutant and wild type – multiple mutant alleles needed to be convincing
• Complement mutation by making a transgenic with the wild type copy of the candidate gene.
Finding a gene based on phenotype
• 1. 100’s of DNA markers mapped onto each chromosome – high density linkage map.
• 2. identify markers linked to trait of interest by recombination analysis
• 3. Narrow region down to a manageable length of DNA – for cloning and sequence comparison
• 4. Compare mutant and wild type sequences to find differences that could cause mutant phenotype
• 5. Prove that mutation is responsible for phenotype.
